RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 507–511

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2333

UV photodissociation of phospho-seryl-containing

peptides: laser stabilization of the phospho-seryl bond

with multistage mass spectrometry

Jerome Lemoine1*, Thibault Tabarin2, Rodolphe Antoine2, Michel Broyer2

and Philippe Dugourd1

1Sciences Analytiques, UMR 5180 (Universite Lyon I et CNRS), 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France2Laboratoire de Spectrometrie Ionique et Moleculaire, UMR 5579 (Universite Lyon I et CNRS), 43 Bd du 11 Novembre 1918, 69622 Villeurbanne

cedex, France

Received 29 September 2005; Revised 2 December 2005; Accepted 3 December 2005

Protonated precursor ions of phosphorylated peptides containing a tyrosyl residue have been sub-

jected to UV laser-induced dissociation (LID) at a wavelength of 220nm and to collision-induced

dissociation (CID) in an ion trap. As expected, neutral loss of the phosphate group is one of the

predominant fragmentation channels during CID together with H2O elimination. In contrast,

LID leads mainly to the homolytic cleavage of the tyrosyl side chain and a restrained loss of the

phosphate group. Interestingly, the intensity of the dephosphorylated fragment ion is greatly mini-

mized when CID is carried out next on the radical precursor ion of the singly and doubly charged

species. Copyright # 2006 John Wiley & Sons, Ltd.

Exhaustive characterization of the post-translational events

that affect proteins is of fundamental importance to fully

establish the relationship between the protein activity and

the state and nature of these modifications. Indeed, the cata-

lytic properties, the subcellular localization, the turnover or

interactionswith other proteins aremost often under the con-

trol of switches governed either byproteolysis or the addition

of chemical groups on the amino acid side chains. Among

them, phosphorylation is a ubiquitous process that plays a

crucial role inmost of the essential biological events,1 i.e. tran-

scription, signal transduction, or apoptosis. In thefieldofpro-

teomics, protein identification is now routinely achieved by

mass spectrometry combined with bioinformatics using

mass fingerprinting of peptides obtained by trypsin hydroly-

sis, or partial peptide sequences deduced from tandemmass

spectrometry (MS2) experiments. When protonated precur-

sor ions of phosphorylated peptides are selected, collision-

induced dissociation (CID)-MS2 spectra exhibit intense ions

originating from gas-phase elimination of phosphate ester

(HPO3,�80Da) or phosphoric acid (H3PO4,�98Da).2 While

these characteristic neutral losses highlight the phosphory-

lated peptides, the localization on the peptide backbone of

the amino acid residues bearing the phosphate moiety often

fails as informative fragments ions are usually of low inten-

sity, or lacking. Electron capture dissociation3 (ECD), or

more recently electron transfer dissociation4 (ETD), have

emerged as powerful ‘mild’ dissociation techniques that do

not induce fragmentation of labile bonds involved between

conjugated groups and peptides. Basically, the reduced

cation species [MþnH](n�1)þ dissociates, leading to fragment

ions of the c and z series. The interest in ECD has now been

widely illustrated for unambiguous assignment of phosphor-

ylation,5O-glycosylation,6 or g-carboxylation sites,7 aswell as

for intact protein sequencing in a top-down approach.8 As an

alternative, a straightforward positioning of phosphate or

glycan moieties was also obtained by CID-MS2, or post-

source decay analysis, of the singly charged cation generated

by matrix-assisted laser desorption/ionization (MALDI) of

peptides derivatized at their N-terminus with a phospho-

nium group.9 In this case, the apparent stability of the conju-

gatedmoiety is likelydue to the lackof amobile proton able to

drive a charge-induced fragmentation mechanism.

Recently, Thompson et al. have reported on the interest in

laser-induced dissociation at 157 nm of protonated peptides

generated by MALDI or electrospray ionization (ESI).10 The

157 nm light yields prominent x- or a-type ions (depending

on the location of the basic residue), a typical feature

encountered during high-energy CID, providing a singly

charged precursor ion is selected. Choosing the doubly

charged species, the fragmentation pattern is dominated by b

and y fragment ions, so rather qualitatively similar to the

distribution observed in CID mode. Excitation at higher UV

wavelengths leads to a competition between classical y and b

fragmentation channels and specific channels like side-chain

cleavage of aromatic residues. y and b ions are likely

produced after internal vibrational relaxation (IVR), in a

way similar to low-energy CID. The specific channels may

result from direct dissociation in electronic excited states or

after relaxation in the ground state but prior to IVR. The

recent study by Oh et al.,11 dealing with laser-induced

dissociation (LID) at 266 nm of 4-sulfophenyl isothiocyanate

Copyright # 2006 John Wiley & Sons, Ltd.

*Correspondence to: J. Lemoine, Sciences Analytiques, UMR 5180(Universite Lyon I et CNRS), 43 Bd du 11 Novembre 1918,69622 Villeurbanne cedex, France.E-mail: jerome.lemoine@univ-lyon1.frContract/grant sponsor: CNRS and Ezus Lyon 1.

derivatives of peptides, confirms the differences between the

excitation mechanisms in UV LID and CID. Instead of a

unique and predictable y fragment series as expected from

the work of Keough et al.,12 the tandem mass spectra

exhibited, in addition, strong signals attributed to w, v ions

aswell as intense internal fragments.We recently studied the

laser wavelength dependence of UV photodissociation of

peptides showing the opening of numerous and specific

channels at 220 nm.13 In the study reported here, we

investigated the dissociation behavior of singly and doubly

protonated ions of phosphorylated peptides during a laser

irradiation at 220 nm, then followed by a CID experiment.

The results obtained with two models of tyrosyl-containing

phosphorylated peptides show that LID might constitute a

promising tool to stabilize the phospho-peptide bond.

EXPERIMENTAL

InstrumentationMass spectrometry was performed using a modified com-

mercial ion trap mass spectrometer (LCQ DUO with the

MSn option; Thermo Electron, San Jose, CA, USA) equipped

with an off-axis ESI source. The ring electrode of the quadru-

pole ion trap was drilled to allow introduction of a UV laser

beam (see Tabarin et al.13 for details). The laser is a nanose-

cond frequency-doubled tuneable OPO laser pumped by an

Nd3þ:YAG laser operated at a repetition rate of 20Hz. Before

entering the trap, the laser beam goes through a mechanical

shutter that is electronically synchronized with the mass

spectrometer and is collimatedwith two 2-mmdiameter pin-

holes. A cylindrical lens (f¼ 500mm) located �500mm from

the center of the trap is used to correct the divergence of the

laser beam that occurs in one dimension. The following pro-

cedure was used to perform LID/CID-MS3 experiments. Sin-

gly or doubly charged precursor ions are first isolated in the

trap. After isolation, they are irradiated with the laser during

10 s (200 laser shots). The laser power monitored by a photon

detector is kept below 100mJ per pulse, and l¼ 220 nm was

used for all the results reported here. After irradiation, a sin-

gle photofragment is isolated in the trap. These selected ions

are fragmentedbyCIDusinghelium (purity > 99.9999vol.%)

as collision and damping gas in the trap. The ions are finally

ejected from the trap and the resulting mass spectrum is

recorded. MS2- and MS3-CID experiments were performed

using the same experimental set-up. For all experiments, a

constant number of ions was maintained inside the trap

(�600). The same collision activation energy was used for

CID and LID/CID experiments.

Sample preparationThe two synthetic phosphorylated peptides, peptide 1

YSDPpSSTST and peptide 2 Ac-VYKpSPVVSGDTSPRHL-

amide, were infused in the ESI source at a concentration of

60 mM in 1% acid acetic containing 1:1 H2O/CH3OH (v/v).

RESULTS AND DISCUSSION

Singly protonated YSDPpSSTSTFull-scan ESI of phosphorylated peptide 1 consists mainly of

the singly charged [MþH]þ molecular ion at m/z 1023. The

subsequent CID-MS2 spectrum is given in Fig. 1(a) and exhi-

bits prominent fragments originating from the direct neutral

loss of one and twowater molecules as well as from the elim-

ination of phosphoric acid (loss of 98Da). Fragmentation of

the peptide backbone yields mainly ions of the b series,

namely b6 (m/z 717), b7 (m/z 818), and b8 (m/z 905). A satellite

peak labelled bD is associatedwith each of them, correspond-

ing to their dephosphorylated counterpart. Phosphorylated

peptide 1 contains one chromophore group (tyrosyl side

chain) that absorbs UV light. The LID spectrum at

l¼ 220 nm of the [MþH]þ molecular ion (Fig. 1(b)) shows a

main fragmentation channel revealed by the intense ion

peak, labelled [MYþH]þ, at m/z 915. This peak arises from

the elimination of the tyrosine side chain plus one hydrogen

loss, i.e. a neutral loss of 108Da. Previously described by

Tabarin et al.13 for leucine enkephalin (YGGFL), this peak

could result from a rearrangement of the transient radical

Figure 1. (a) CID and (b) LID fragment ion spectra of

protonated phosphorylated peptide 1 YSDPpSSTST (m/z

1023 was isolated in the trap before dissociation). For the CID

experiment, an activation time of 30ms with an amplitude

activation adjusted for a 60% attenuation of the precursor ion

beam was used. For the LID experiment, l¼ 220 nm, and the

irradiation time was 10 s (200 laser pulses). (c) LID/CID-MS3

spectrum of the [MYþH]þ fragment ion at m/z 915. Proto-

nated phosphorylated peptide 1 was first fragmented by LID

(l¼ 220 nm, and irradiation time 10 s), and then the m/z 915

ion was isolated. The second stage of fragmentation of this

ion was produced by CID (activation time 30ms, 60%

precursor ion beam attenuation). D-labelled ions correspond

to fragments that have lost phosphoric acid.

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 507–511

508 J. Lemoine et al.

ion occurring by hydrogen loss from the protonated N-

terminus leading to an imminium ion. Direct loss of

hydrogen from the precursor ion is also noticeable and is

characteristic of LID.14,15 Finally, themost striking difference

with the CID spectrum is the lack of phosphate group elimi-

nation. Both the phosphoric acid neutral loss and a, b or y

fragmentation channels seem to be quenched by the Ca–Cb

bond breaking leading to the loss of the aromatic side chain.

This quenching might be explained by the fact that the UV

photon is absorbed by the tyrosyl group which induces a

rapid Ca–Cb bond breaking (close to the absorption) prior

to the IVR mechanism.

Figure 1(c) shows the LID/CID-MS3 spectrum of the

[MYþH]þ (m/z 915) precursor ion. Whereas the fragmenta-

tion pattern is qualitatively similar in terms of distribution of

the fragment ions (a similar series of b6, b7, b8 and y is found)

to the one obtained from the CID experiment (Fig. 1(a)), a

significant difference lies in the events leading to the

fragmentation of the bond between the phosphate group

and the seryl residue.As illustrated by thehistogram inFig. 2,

the relative abundance of ions issued either from the direct or

consecutive (bD ions series) loss of phosphoric acid is greatly

minimized when CID is carried out on the [MYþH]þ

precursor ion prepared by LID. A possible explanation is a

partial absorption of the UV light via the carbonyl group of

the peptide bond occurring during the LID stage, which

would increase the internal energy of the peptide backbone

prior to the CID stage. The lack of efficient IVR would then

promote the breaking of the peptide bonds at the expense of

the more energetically favored phosphate side-chain

removal.

Singly and doubly protonatedAc-VYKpSPVVSGDTSPRHL-amideThe N-terminus acetylated and C-terminus amidated form of

peptide 2 (Ac-VYKpSPVVSGDTSPRHL-amide) was used to

further explore the fragmentation pathway of phosphorylated

peptides during LID and LID/CID-MS2. Electrospraying this

compound leads mainly to singly and doubly charged

molecular ions at m/z 1863 and 932, respectively. The

CID-MS2 spectrum of singly protonated peptide 2 (Fig. 3(a))

Figure 2. (a) Relative abundances of phosphate-retaining

fragment ions (P

b; y ) as compared to fragment ions that

have lost phosphoric acid (P

Frag�) during CID of singly

protonated peptide 1 (m/z 1023), singly protonated peptide 2

(m/z 1863), and doubly protonated peptide 2 (m/z 932).

(b) Relative abundances of phosphate-retaining fragment ions

(P

bY ; yY ;P

yY ;P

bY ; yY ) as compared to fragment ions

that have lost phosphoric acid (P

FragY ;�) during LID/CID-

MS3 of singly protonated peptide 1 (CID of [MYþH]þ, m/z

915), singly protonated peptide 2 (CID of [MYþH]þ.,m/z 1756)

and doubly protonated peptide 2 (CID of [MYþ2]2þ.,m/z 878).

Figure 3. (a) CID and (b) LID fragment ion spectra

of singly protonated phosphorylated peptide 2 Ac-

VYKpSPVVSGDTSPRHL-amide (m/z 1863 was isolated in

the trap before dissociation). For the CID experiment, an

activation time of 100ms with an amplitude activation

adjusted for a 60% attenuation of the precursor ion beam

was used. For the LID experiment, l¼ 220nm, and the

irradiation time was 10 s (200 laser pulses). (c) LID/CID-MS3

spectrum of the [MYþH]þ. fragment ion (m/z 1756). Proto-

nated phosphorylated peptide 2 was first fragmented by LID

(l¼ 220 nm, and irradiation time 10 s), and then them/z 1756

ion was isolated. The second stage of fragmentation of this

ion was produced by CID (activation time 100ms, 60%

precursor ion beam attenuation).

UV photodissociation of phospho-seryl-containing peptides 509

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 507–511

displays two intense peaks attributed to H2O and H3PO4

neutral losses. Compared topeptide 1, a rather complete series

of phosphorylated b (12, 14, 15) and y (5, 6, 7, 8, 9, 10, 12, 13, 14,

15) fragments furnish enough sequence information to deline-

ate the phosphorylation site on serine 4.

Subjecting the same protonated ions to LID leads to side-

chain cleavage of the tyrosyl residue (Fig. 3(b)), although in a

less prominent manner than in the case of peptide 1.

Concomitantly, neutral losses of 28, 44 and 98Da (phosphoric

acid loss) are observed. Moreover, after the side-chain

cleavage, no rearrangement of the radical ionic site occurs

as revealed by the [MYþH]þ. fragment ion observed 107Da

below the precursor ion instead of 108Da as in the case of

peptide 1. This might be explained by the acetylated state of

the N-terminus which would inhibit the formation of an

imminium ion or by the position of the tyrosil group located

further from the N-terminus than in peptide 1. Then, the

radical [MYþH]þ. ion was subjected to a LID/CID-MS3

experiment (Fig. 3(c)).A comparisonof the relative intensities

of the peaks (Fig. 2) attributed to direct or consecutive losses

of the phosphate group shows a stabilization of the

phosphate–seryl bond in the radical precursor ion prepared

by LID compared to CID carried out on the native peptide.

This feature is qualitatively similar to the one obtained with

the even-electron species generated during LID carried on

peptide 1.

Overall, the most striking differences were observedwhen

comparing the fragmentation pattern of the doubly proto-

nated species during CID-MS2 and LID/CID-MS3. In CID

mode (Fig. 4(a)), the doubly protonated native peptide

initiates two major neutral losses, i.e. H2O (m/z 923) and

H3PO4 (m/z 883), aswell as cleavages of the peptide backbone

of the b- and y-type series.

Two doubly charged fragment peaks at m/z 918 and 878,

attributed respectively to neutral losses of 28 and 107Da, are

the main species observed in the LID mass spectrum of the

Figure 4. (a) CID and (b) LID fragment ion spectra of doubly protonated phosphory-

lated peptide 2 Ac-VYKpSPVVSGDTSPRHL-amide (m/z 932 was isolated in the trap

before dissociation). For the CID experiment, an activation time of 200ms with an

amplitude activation adjusted for 60% attenuation of the precursor ion beam was used.

For the LID experiment, l¼ 220nm, and irradiation time was 10 s (200 laser pulses).

(c) LID/CID-MS3 spectrum of the [MYþ2H]2þ. fragment ion (m/z 878). Protonated

phosphorylated peptide 2 was first fragmented by LID (l¼ 220nm, and irradiation time

10 s), and then the m/z 878 ions were isolated. The second stage of fragmentation of

this ion was produced by CID (activation time 100ms, 60% precursor ion beam

attenuation).

510 J. Lemoine et al.

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 507–511

doubly protonated precursor ion (Fig. 4(b)). The main

fragment corresponds likely to the elimination of a CO group

and has been previously observed during LID. The second

peak is attributed to the side-chain cleavage of the tyrosine

leading to the formation of a radical ionic moiety.

The LID/CID-MS3 fragment ion pattern of this radical

[MYþ2H]2þ cation (Fig. 4(c)) underlines not only a striking

contrast of the dissociation behavior compared to the one of

the native peptide observed during CID, but also to the

comportment of the singly charged radical [MYþH]þ. species.

Indeed, the direct loss of the phosphate group is here fully

inhibited. Moreover, no secondary loss of this moiety from

the backbone cleavages, dominated by b and a species, is

detectable (Fig. 2). Satellite peaks corresponding to neutral

losses of 16 and 44Da from the two series of b and a fragment

ions are systematically observed and account for the large

density of peaks. They may originate from sequential losses

of the methyl moiety and of the acetyl groups from the

acetylated peptide N-terminus. Interestingly, no charged

C-terminal fragment of the y-type is detected. Due to the

presence of an expected mobile charge, one would have

predicted for this doubly protonated precursor ion a

fragmentation pattern similar to CID. Since the LID/CID-

MS3 spectrum of the [MYþ2H]2þ. precursor ion is fundamen-

tally different, a prominent influence of the radical site

on the fragmentation process may be advanced, as recently

observed in the fragmentationof free radical initiator-peptide

conjugates.16 However, any attempt to propose a supporting

mechanism would be speculative at this time.

CONCLUSIONS

Protonated ions of phosphorylated peptides at seryl residues

have been subjected to dissociation by CID-MS2, LID-MS2

and LID/CID-MS3. During CID-MS2 on the singly and dou-

bly charged ions, the classical neutral loss of phosphoric acid

is predominant. This loss is strongly restrained in LID-MS2.

The LID experiment induces mainly the homolytic cleavage

of the aromatic side chain of the tyrosyl residues. Another

striking and unexpected fragmentation behavior observed

between direct CID-MS2 and CID/LID-MS3 concerns the

neutral loss of the phosphate group. This loss is partially

inhibited when LID/CID-MS3 experiments are carried out

on singly protonated ions. It is almost totally suppressed by

selecting the doubly charged precursor ions. Whether such

dissociation features might be extended to any phosphory-

lated peptide sequence on seryl or threonyl residues has to

be evaluated with a wider set of peptide sequences. Further-

more, the influence of the phosphorylation site localization

on the dissociation pattern should obviously be carefully

explored. So far, the mechanisms bywhich odd-electron pre-

cursor ions, or the even species issued from hydrogen rear-

rangement, dissociate during LID/CID experiments remain

to be elucidated. Nonetheless, these preliminary results

suggest LID as a newpromising route aimed at phosphoryla-

tion site localization and require other wavelengths to be

investigated.

AcknowledgementsThe authors wish to thank Dr. Richard Munton (Universitat

Zurich) for providing the phosphorylated peptides, F. O.

Talbot for experimental help at the beginning of this work,

and CNRS and Ezus Lyon 1 for their financial support.

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UV photodissociation of phospho-seryl-containing peptides 511

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 507–511